WO2018006881A1 - Recombinant immune-checkpoint receptor and application thereof - Google Patents

Recombinant immune-checkpoint receptor and application thereof

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Publication number
WO2018006881A1
WO2018006881A1 PCT/CN2017/092377 CN2017092377W WO2018006881A1 WO 2018006881 A1 WO2018006881 A1 WO 2018006881A1 CN 2017092377 W CN2017092377 W CN 2017092377W WO 2018006881 A1 WO2018006881 A1 WO 2018006881A1
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nucleic acid
lymphocytes
encoding
construct
according
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PCT/CN2017/092377
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French (fr)
Chinese (zh)
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陈思毅
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生命序有限公司
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14Blood; Artificial blood
    • A61K35/17Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K19/00Hybrid peptides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62DNA sequences coding for fusion proteins
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10Cells modified by introduction of foreign genetic material Not used, see subgroups

Abstract

A recombinant receptor and an application thereof. The recombinant receptor comprises: a cellular immune-checkpoint molecular segment; an immune stimulator molecular segment; and a T-cell receptor zeta chain.

Description

Immune Receptor Recombinant Its Application Checkpoint

Priority Information

This application claims filed with the Chinese State Intellectual Property Office July 8, 2016, patent application number and priority interests 201,610,538,767.9 patent applications, and by reference in its entirety here.

FIELD

The present invention relates to the field of biomedicine, in particular, the present invention relates to a recombinant immune checkpoint receptors and their applications, and more particularly, the present invention relates to a recombinant receptor, a nucleic acid, transfected lymphocytes, construct, transgenic lymphocytes, treatment of cancer therapeutic compositions and methods of lymphocyte immune killing ability.

Background technique

Cancer, since the inner cell gene mutation leads to a disease of uncontrolled cell proliferation. Has become a major threat to human health is one of the main causes of death. World Health Organization (WHO) published in the "Global Cancer Report 2014" that the 2012 global cases and deaths in patients with cancer are increasing rapidly, while nearly half of new cancer cases occur in Asia, most of them in China, China new cancer cases highest in the first. "2012 Annual Report tumor registration China" Data show that China's annual increase of about 3.5 million cancer cases, about 2.5 million people died. Thus, cancer treatments seek high specificity is of great clinical value.

Traditional cancer treatments including surgery, radiotherapy and chemotherapy, but several methods have great limitations, such as cancer cell invasion due proximal or distal metastasis, tumor metastasis after surgical resection of a high recurrence rate, the radiation and chemotherapy for the body's own normal cells, especially hematopoietic system and immune system can cause serious damage, so for patients with tumor metastasis has occurred it is difficult to achieve a better long-term efficacy. With the further development of in-depth research and biotechnology molecular mechanisms of cancer, targeted therapy and immunotherapy plays an increasing role in the comprehensive treatment of tumors. Targeted therapies including monoclonal antibodies (sometimes classified as passive cell transfusion and cancer vaccines. Immunotherapy by mobilizing the body's immune system and the tumor microenvironment to enhance the anti-tumor immunotherapy free) and small molecular targeted drugs, immunotherapy including cytokine therapy, monoclonal antibody immunoassay point, adoptive immunotherapy, to control and kill tumor cells, and therefore high efficiency, specificity, good tolerability advantages, has broad prospects in tumor treatment.

However, tumor immunotherapy, remains to be further research and development to enhance clinical efficacy.

SUMMARY

The present invention is intended to solve at least one problem related art technology to some extent. To this end, an object of the present invention is to provide a recombinant receptor and its effective utilization method of enhancing an immune lymphocytes to kill tumor cells.

In a first aspect of the present invention, the present invention provides a recombinant receptor. According to embodiments of the present invention, the recombinant receptor comprising: a cellular immune checkpoint molecule fragment; immunostimulatory molecule fragments; and T cell receptor zeta chain. According to an embodiment of the present invention, lymphocytes expressing recombinant receptors embodiment of the present invention, can effectively enhance the specific killing effect on tumor cell lymphocytes.

According to an embodiment of the present invention, the recombinant receptor may further comprise at least one of the following additional features:

According to an embodiment of the present invention, the cellular immune checkpoint molecule PD1. PD1 may be combined with a tumor cell specific expression of the PD-L1 or PD-L2. Furthermore, lymphocytes express recombinant receptors of the present embodiment of the invention, its tumor cells, particularly targeting of anti-PD-L1 or PD-L2-positive tumor cells is further enhanced.

According to an embodiment of the present invention, the cellular immune checkpoint molecular fragment comprising an extracellular region of the PD1 of the transmembrane region, and optionally, the immunostimulatory molecule comprises a fragment of CD28 intracellular domain of a transmembrane region and optionally . PD1 having the extracellular region of PD-L1 function area or PD-L2 is specifically expressed on tumor cells in combination, with the CD28 intracellular activation of immune stimulation ribbon signaling pathway, cells expressing lymphocytes further embodiment of the present invention recombinant receptors embodiment, the targeting of tumor cell killing effect is further improved.

According to an embodiment of the present invention, the recombinant receptor comprising: (a) PD1 of the extracellular and transmembrane domains; and (b) said intracellular region of CD28, or comprising: (i) the PD1 extracellular region; and (ii) of the CD28 intracellular and transmembrane domains. Both combinations were retained PD1 ribbon and is specifically expressed on tumor cells PD-L1 or PD-L2 combined ribbon and CD28 activation of immune stimulation signal pathway, while the transmembrane region or both of CD28 PD1 transmembrane region, transmembrane receptor can make recombinant expression, lymphocyte expressing a recombinant receptor further embodiment of the present invention is that targeting tumor cell killing effect is further improved.

According to an embodiment of the present invention, the T cell receptor zeta chain CD3zeta chain. Specifically CD3zeta chain T cell receptor activation of downstream signaling pathways, cells expressing a recombinant receptor further embodiment lymphocytes embodiment of the present invention, the killing effect on tumor cells is further improved.

According to an embodiment of the present invention, the C-terminal fragment of the molecule checkpoint immune cells with the immunostimulatory molecule is connected to the N-terminal fragment, the C-terminus of the immunostimulatory molecule fragments with the T cell receptor zeta chain N terminal coupled. Recombinant receptor fragments related embodiment of the present invention in the connecting sequence, facilitate positioning of the relevant fragments in a cell, and thus more conducive to play the corresponding functions - targeting, transmembrane signaling pathways and activation of the immune stimulation of activated T cell receptor signaling pathways, which targeting ability to kill tumor cells is further improved.

In the second aspect of the present invention, the present invention provides a recombinant receptor. According to an embodiment of the present invention, the recombinant receptor having SEQ ID NO: 1 or the amino acid sequence shown in 2.

Figure PCTCN2017092377-appb-000001

Figure PCTCN2017092377-appb-000002

According to an embodiment of the present invention, lymphocytes expressing recombinant receptors embodiment of the present invention, can effectively enhance lymphocytes, especially PD-L1 or PD-L2 specific killing effect on tumor cells positive tumor cells.

In a third aspect of the present invention, the present invention provides a nucleic acid. According to an embodiment of the present invention, the aforementioned recombinant nucleic acid encoding the receptor, optionally, the nucleic acid having SEQ ID NO: 3 or the nucleotide sequence shown 4.

Figure PCTCN2017092377-appb-000003

Figure PCTCN2017092377-appb-000004

The nucleic acid embodiment of the present invention into the recipient embodiment lymphocytes, a recombinant nucleic acid encoding a transmembrane receptor expression in lymphocytes, the lymphocytes specific killing effect on tumor cells was significantly improved.

In a fourth aspect of the present invention, the present invention provides a construct. According to an embodiment of the present invention, the construct carrying the nucleic acid previously described. Examples of the construct into the recipient lymphocytes present invention, nucleic acid material carried construct encoding a recombinant transmembrane receptor expression in lymphocytes, the lymphocytes specific killing effect on tumor cells was significantly improved.

According to an embodiment of the present invention, the construct may further include at least one of the following additional features:

According to an embodiment of the present invention, the construct further carry a nucleic acid encoding a non-functional EGFR, nucleic acid encoding said non-functional EGFR has SEQ ID NO: 5 nucleotide sequence.

Figure PCTCN2017092377-appb-000005

According to an embodiment of the present invention, non-functional EGFR lack N- terminal receptor ligand binding domain and an intracellular receptor tyrosine kinase activity, including wild-type EGFR receptor transmembrane and complete binding of anti-EGFR antibody sequence, so no function as suicide EGFR receptor labeled lymphocytes. Example constructs of the invention into the recipient lymphocytes expressing non-functional EGFR receptor may be on the premise of ensuring effective targeting of tumor cell killing effect of the lymphocytes, if patients had serious adverse events, lymphocytes anti-EGFR antibody is cleared, thereby improving the safety of the present constructs embodiment of the invention the treatment of cancer patients.

According to an embodiment of the present invention, the construct further carries an internal ribosome entry site sequence, the internal ribosome entry site sequence SEQ ID NO: 6 nucleotide sequence, and the internal ribosome EGFR nonfunctional nucleic acids between the entry site of the sequence arranged in front of the nucleic acid and encoded.

Figure PCTCN2017092377-appb-000006

Figure PCTCN2017092377-appb-000007

Introducing an internal ribosome entry site sequence, such that expression of non-functional EGFR initial coding nucleic acid is not dependent on a 5 'cap structure and a nucleic acid encoding the recombinant receptor and a nucleic acid encoding a non-functional EGFR proportional expression, and thus more conducive expression regulation construct the embodiment of the present invention into a recipient lymphocytes embodiment, the safety of the treatment of the obtained transgenic higher lymphocytes.

According to an embodiment of the present invention, the construct further carry a nucleic acid encoding a linker peptide, nucleic acid encoding a linker peptide having SEQ ID NO: nucleotide sequence shown in 7-10, and the nucleic acid encoding a linker peptide arranged in front between the nucleic acids and the nucleic acid encoding said non-functional EGFR.

Figure PCTCN2017092377-appb-000008

According to an embodiment of the present invention, the embodiment of the constructs into the recipient lymphocytes embodiment of the present invention, the encoded peptide can be cut are connected in the lymphocytes, introduction of the linker peptide such that the expressed recombinant receptors and non-functional EGFR expression in a non-confluent lymphocyte membranes.

According to an embodiment of the present invention, the construct further carrying a first promoter, the first nucleic acid promoter operatively linked to the aforementioned, optionally, the first promoter selected from the group comprising CMV, EF-1, LTR or RSV promoter. Regulation of the first promoter can independently activate expression of the first nucleic acid molecule, and thus is more conducive to the expression of a corresponding nucleic acid molecule. The inventors have found, CMV, EF-1, LTR promoter or RSV promoter capable of efficiently expressing a nucleic acid according to the foregoing, the efficiency of expression of the previously described nucleic acid significantly increased.

According to an embodiment of the present invention, the construct further carries a second promoter, said second promoter and said nucleic acid encoding a non-functional EGFR operably linked, optionally, comprise a second promoter selected from since CMV, EF-1, LTR or RSV promoter. Promoter and the second promoter are independently a nucleic acid encoding a non-functional EGFR expression, and thus more conducive to expression control nucleic acid encoding a non-functional EGFR. The inventors have found, CMV, EF-1, LTR promoter or RSV promoter capable of efficiently expressing a nucleic acid encoding a non-functional EGFR, expression efficiency of nucleic acid encoding a non-functional EGFR significantly increased.

According to an embodiment of the present invention, the vector construct is a retroviral vector, lentiviral vector or an adeno-associated virus vector. Vector described above may be implemented in a high expression of the receptor nucleic acid-carrying cells, high therapeutic efficiency.

In a fifth aspect of the present invention, the present invention provides a construct. According to an embodiment of the present invention, the construct carrying the nucleic acid molecule of the following: a nucleic acid molecule (a) encoding a fragment of such molecule immune checkpoint, the checkpoint immune molecule fragment having SEQ ID NO: 11 amino acid sequence shown in FIG. 12, the nucleic acid molecule encodes an immune checkpoint molecule fragments having SEQ ID NO: 14 or the nucleotide sequence of FIG. 13; (2) a nucleic acid molecule encoding an immunostimulatory molecule fragments, said immunostimulatory molecule fragment having SEQ ID NO: the amino acid sequence shown in 1516, the immunostimulatory molecule is a nucleic acid molecule encoding a fragment of SEQ ID NO: 17 or the nucleotide sequence as shown in 18; and nucleic acid molecules (3) encoding a T cell receptor zeta chain, the T cell receptor zeta chain having SEQ ID NO: 19 amino acid sequence, a nucleic acid molecule encoding the T cell receptor zeta chain having SEQ ID NO: 20 nucleotide sequence.

Figure PCTCN2017092377-appb-000009

Figure PCTCN2017092377-appb-000010

Figure PCTCN2017092377-appb-000011

Examples of the construct into the recipient lymphocytes present invention, molecular fragment thereof encoded by a nucleic acid carried by a fusion construct consisting of receptor protein, and the transmembrane expressed in lymphocytes, the lymphocytes specific for tumor cells killing effect of significantly improved.

According to an embodiment of the present invention, the construct may further include at least one of the following additional features:

According to an embodiment of the present invention, the construct further carrying nucleic acid molecule encoding a non-functional EGFR, a nucleic acid molecule encoding a non-functional EGFR having the SEQ ID NO: 5 nucleotide sequence shown, having a non-functional EGFR SEQ ID NO: 21 amino acid sequence shown.

Figure PCTCN2017092377-appb-000012

As described above, embodiments of the construct into the recipient of the present invention lymphocytes expressing non-functional EGFR receptor may be on the premise of ensuring effective targeting of tumor cell killing effect of the lymphocytes, if the patient serious adverse the reaction, lymphocytes may be anti-EGFR antibody clearance, thereby improving the safety of the treatment of cancer patients constructs embodiment of the present invention, and lymphocytes.

According to an embodiment of the present invention, the nucleic acid molecule further carries an internal ribosome entry site sequence construct, entry site sequence of a nucleic acid molecule having the internal ribosome SEQ ID NO: 6 nucleotide sequence, and between the encoded internal ribosome entry site sequence in said nucleic acid molecule is provided encoding a T cell receptor zeta chain and a nucleic acid molecule encoding a non-functional EGFR said nucleic acid molecule. As described above, the introduction of internal ribosome entry site sequence is more conducive to expression control, the safety of the treatment of the obtained transgenic higher lymphocytes.

According to an embodiment of the present invention, the construct further carry a nucleic acid encoding a linker peptide, the nucleic acid encoding a linker peptide SEQ ID NO: 7 nucleotide sequence shown in ~ 10, the connecting peptide has SEQ ID NO the amino acid sequence between 22 to 25 and nucleic acid encoding a linker peptide is provided and said nucleic acid molecule encoding said encoding T cell receptor zeta-chain non-functional EGFR nucleic acid molecule:

Figure PCTCN2017092377-appb-000013

Figure PCTCN2017092377-appb-000014

Construct described above, the embodiment of the present invention into the recipient-lymphocytes, the encoded peptide can be cut are connected in the lymphocytes, the introduction of such connecting peptide molecular fragment consisting of the expressed recombinant receptor and non-functional EGFR expression in a non-confluent lymphocyte membranes.

According to an embodiment of the present invention, the construct further carrying a first promoter, the first nucleic acid molecule encoding a promoter of the immune checkpoint molecule fragment operably linked, optionally, the first start selected promoters include CMV, EF-1, LTR or RSV promoter. As described above, CMV, EF-1, LTR promoter or RSV promoter can efficiently express nucleic acid molecules encoding the aforementioned fragments immune checkpoint molecule, nucleic acid molecule encoding an immunostimulatory molecule and a fragment encoding T cell receptor zeta chain nucleic acid molecule, the expression efficiency of nucleic acid molecule fragments of the foregoing significantly increased.

According to an embodiment of the present invention, the construct further carries a second promoter, said second promoter and said nucleic acid encoding a non-functional EGFR molecule is operably linked, optionally, a second promoter comprising selected, CMV, EF-1, LTR or RSV promoter. As described above, CMV, EF-1, LTR promoter or RSV promoter capable of efficiently expressing the nucleic acid molecule encoding a non-functional EGFR expression efficiency of nucleic acid molecule encoding a non-functional EGFR significantly increased.

According to an embodiment of the present invention, the vector construct is a retroviral vector, lentiviral vector or an adeno-associated virus vector. Vector described above may be implemented in a high expression of the receptor nucleic acid-carrying cells, high therapeutic efficiency.

In a sixth aspect of the present invention, the present invention provides a transgenic lymphocytes. According to an embodiment of the present invention, the transgenic lymphocytes expressing recombinant receptors previously described, optionally, said transgenic non-functional lymphocytes express EGFR. Transgenic embodiment of the present invention lymphocytes strong specific killing effect on tumor cells, safety.

According to an embodiment of the present invention, the transfected lymphocytes may further comprise at least one of the following additional features:

According to an embodiment of the present invention, the lymphocyte antigen specific T lymphocytes, optionally, the tumor infiltrating lymphocytes are T-lymphocytes, optionally, the lymphocyte is a T lymphocyte, any Alternatively, the lymphocyte is a Natural killer T lymphocytes, optionally, the natural killer cells are lymphocytes. The antigen-specific T lymphocytes embodiment of the present invention, tumor-infiltrating T lymphocytes, peripheral blood T lymphocytes, natural killer T lymphocytes or natural killer cells, tumor cells can be achieved specific immune destruction, high safety.

In a seventh aspect of the present invention, the present invention provides a process for preparing the foregoing transgenic lymphocytes. According to an embodiment of the present invention, the method comprising: the aforementioned constructs into lymphocytes or T lymphocytes. With the above-described method embodiments of the present invention, it can be easily and efficiently obtain transgenic lymphocytes previously described, as described above, the obtained transgenic significantly improve the specific killing of lymphocytes to tumor cells, safety.

In an eighth aspect of the present invention, the present invention provides a therapeutic composition for the treatment of cancer. The implementation of the embodiment of the present invention, the therapeutic composition comprising: a recombinant receptor according to the foregoing, the previously described nucleic acid, the previously described transgenic construct or in front of the lymphocytes. With the embodiment of the therapeutic composition of the present invention enables efficient, safe killing of tumor cells.

In a ninth aspect of the present invention, the present invention provides a method for increasing the ability of anti-lymphocyte immune therapy. According to an embodiment of the present invention, the method comprising: making the lymphocytes express recombinant receptors previously described. With the above-described embodiment of the method of the present invention can effectively improve the anti-lymphocyte specific immunity to tumor cells.

In a tenth aspect of the present invention, the present invention provides a method of treating cancer. According to an embodiment of the present invention, the method comprising: administering to a patient in front of the recombinant receptor, the nucleic acid according to the foregoing, the previously described transgenic construct or in front of the lymphocytes. With the method of treatment of the present invention, an embodiment can be realized on tumor cells in vivo tumor patients efficiently and safely killing.

BRIEF DESCRIPTION

FIG 1 is a schematic structural diagram of an embodiment of the viral vector according to the present invention is slow;

Figure 2 is the results of FIG PD1-CD28-CD3zeta recombinant receptors and non-functional EGFR receptor is an anti-EGFR antibody lymphocytes mediating ADCC killing clearance coexpression embodiment of the present invention; and

FIG. 3 shows the results of FIG PD1-CD28-CD3zeta recombinant receptors and non-functional EGFR receptor anti-PD-L1 lymphocyte positive tumor cells co-expressed in accordance with an embodiment of the present invention.

detailed description

Embodiments of the present invention is described in detail. The embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention.

Incidentally, the terms "first", "second" are used to indicate or imply relative number of features of importance or implicitly specify the indicated purpose of description and should not be understood. Thus, there is defined "first", "second" features may be explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "more" is two or more.

Recombinant receptor protein

In one aspect, the present invention provides a recombinant receptor. According to an embodiment of the present invention, the recombinant receptor comprising: a cellular immune checkpoint molecule fragment; immunostimulatory molecule fragments; and T cell receptor zeta chain. According to an embodiment of the present invention, lymphocytes expressing recombinant receptors embodiment of the present invention, can effectively enhance the specific killing effect on tumor cell lymphocytes.

According to a particular embodiment of the present invention, the cellular immune checkpoint molecule PD1. PD1 may be combined with a tumor cell specific expression of the PD-L1 or PD-L2, and further, lymphocytes expressing recombinant receptors embodiment of the present invention, under the guidance of PD1 lymphocytes, specifically target tumor cells, which of target tumor cells is further enhanced.

According to another embodiment of the present invention, a particular embodiment, the immune cell checkpoint molecular fragment comprising an extracellular region PD1 and optionally the transmembrane domain, a fragment comprising an immunostimulatory molecule CD28 intracellular domain, and optionally the transmembrane region. For example, according to embodiments of the present invention, the recombinant receptor may comprise: (a) PD1 extracellular and transmembrane domains; and intracellular region (b) CD28, or comprising: the extracellular domain (i) PD1 of; and (ii) CD28 intracellular and transmembrane domains. PD1 having the extracellular region of PD-L1 function area or PD-L2 is specifically expressed on tumor cells in combination, with the CD28 intracellular signaling pathways activated immunostimulatory ribbon, while the transmembrane region or both PD1 CD28 transmembrane region, transmembrane receptor can make recombinant expression, lymphocyte expressing a recombinant receptor further embodiment of the present invention is that targeting tumor cell killing effect is further improved.

Further, according to a further embodiment of the present invention, T cell receptor zeta chain CD3zeta chain. CD3zeta associated chain T-cell receptor (TCR) signaling pathway, the chain CD3zeta triggered, the Zeta chain may be referred to the same cytoplasmic zeta chain-associated protein 70 (ZAP-70) in combination, ZAP-70 as having an in cytoplasm tyrosine kinase (a PTK) activity of the signal protein, containing two SH-2 (srchomology region 2, SH-2) domain, ZAP-70 and SH-2 molecule zeta chain phosphorylated tyrosine residues combined, ZAP-70 activation may further activate Ras proteins, and thus the final activated lymphocytes. CD3zeta chain may be T cell receptor-specific activation of downstream signaling pathways, and thus lymphocytes express the recombinant protein of the present embodiment of the invention, the immunostimulatory molecule activation in the functional fragments, and synergy CD3zeta chain activation, the killing effect on tumor cells Further improve.

Finally, according to an embodiment of the present invention, the recombinant proteins in connection order of the corresponding molecule fragments may be: coupled to the N-terminus C-terminus of immune checkpoint molecule fragments cellular immunostimulatory molecule fragments, immunostimulatory molecule fragments C-terminus of T connected to the N-terminal cell receptor zeta chain. The inventors have found that the recombinant protein fragment related embodiment of the present invention in the connecting sequence, facilitate positioning of the relevant fragments in a cell, and thus more conducive to play the corresponding functions - targeting, transmembrane signaling pathways activated immunostimulatory and activating T cell receptor signaling pathway, targeting its ability to kill tumor cells is further improved.

In particular, according to an embodiment of the present invention, the recombinant receptor has the SEQ ID NO: 1 or the amino acid sequence shown in 2. Wherein, SEQ ID NO: 1 represents human PD1 comprising the extracellular region and the transmembrane region, CD28 intracellular and CD3zeta chain recombinant receptor (PD1-ECD-TM-CD28-ICD-CD3zeta) amino acid sequence; SEQ ID NO : 2 represents PD1 comprising human extracellular region, transmembrane region and the amino acid sequence of CD28 and intracellular recombinant receptor CD3zeta chain (PD1-ECD-CD28-TM-ICD-CD3zeta) a. According to an embodiment of the present invention, having the above-described recombinant receptor amino acid sequence, for expression in lymphocytes, can effectively enhance the specific killing effect on tumor cell lymphocytes.

Nucleic acid

Another aspect, the present invention provides a nucleic acid. According to an embodiment of the present invention, the nucleic acid encoding a recombinant receptor according to the foregoing, optionally, the nucleic acid having SEQ ID NO: 3 or the nucleotide sequence shown 4. Wherein, SEQ ID NO: 3 is a nucleotide sequence encoding human PD1 shown comprising an extracellular region and transmembrane region, CD28 intracellular recombinant receptor and CD3zeta chain (PD1-ECD-TM-CD28-ICD-CD3zeta) , SEQ ID NO: 4 encoded by a nucleotide sequence comprising the extracellular domain of human PD1, CD28 transmembrane region and the intracellular region and a recombinant receptor CD3zeta chain (PD1-ECD-CD28-TM-ICD-CD3zeta). The nucleic acid embodiment of the present invention into the recipient embodiment lymphocytes, a recombinant nucleic acid encoding a transmembrane receptor expression in lymphocytes, the lymphocytes specific killing effect on tumor cells was significantly improved.

Construct

Another aspect, the present invention provides a construct. According to an embodiment of the present invention, the construct carrying the nucleic acid previously described. Examples of the construct into the recipient lymphocytes present invention, nucleic acid material carried construct encoding a recombinant transmembrane receptor expression in lymphocytes, the lymphocytes specific killing effect on tumor cells was significantly improved.

Alternatively, according to embodiments of the present invention, the construct carrying the nucleic acid molecule of the following: a nucleic acid molecule (a) encoding a fragment of such molecule immune checkpoint, the checkpoint immune molecule fragment having SEQ ID NO: or an amino acid represented by 1112 sequence, said encoding immune checkpoint molecule fragments having a nucleic acid molecule SEQ ID NO: or a nucleotide sequence as shown in 1314; wherein, SEQ ID NO: 11 amino acid sequence of human extracellular and transmembrane PD1 the amino acid sequence (PD1-ECD-TM) region, SEQ ID NO: 12 is the amino acid sequence shown in the amino acid sequence (PD1-ECD) of the extracellular region of human PD1; SEQ ID NO: 13 encoding the PD1-ECD-TM nucleotide sequence, SEQ ID NO: 14 is the nucleotide sequence encoding the PD1-ECD. Nucleic acid molecule (2) a fragment encoding an immunostimulatory molecule, the immunostimulatory molecule fragment having SEQ ID NO: 15 or the amino acid sequence set forth in 16, said nucleic acid molecule encoding an immunostimulatory molecule fragments having SEQ ID NO: 18. 17 or nucleotide sequence, wherein, SEQ ID NO: 15 is the amino acid sequence shown in the amino acid sequence (CD28-ICD) of the human CD28 intracellular region, SEQ ID NO: 16 amino acid sequence of human CD28 is a transmembrane the amino acid sequence (CD28-TM-ICD) region and the intracellular region, SEQ ID NO: 17 is the nucleotide sequence encoding a CD28-ICD's, SEQ ID NO: 18 is the nucleotide sequence encoding a CD28-TM-ICD; nucleic acid molecules and (3) encoding a T cell receptor zeta chain of the T cell receptor zeta chain having SEQ ID NO: 19 amino acid sequence, the nucleic acid molecule of the T cell receptor zeta chain encoding a polypeptide having the SEQ ID NO: 20 nucleotide sequence. Examples of the construct into the recipient lymphocytes present invention, molecular fragment thereof encoded by a nucleic acid carried by the composition of the constructed recombinant receptor, and the transmembrane expressed in lymphocytes, the lymphocytes specific for tumor cells killing effect significantly.

According to a particular embodiment of the present invention, the nucleic acid further encoding a non-functional EGFR carrying the construct, said nucleic acid encoding a non-functional EGFR having SEQ ID NO: 5 nucleotide sequence. According to an embodiment of the present invention, non-functional EGFR lack N- terminal receptor ligand binding domain and an intracellular receptor tyrosine kinase activity, including wild-type EGFR receptor transmembrane and complete binding of anti-EGFR antibody sequence, so no function as suicide EGFR receptor labeled lymphocytes. Example constructs of the invention into the recipient lymphocytes expressing non-functional EGFR receptor may be on the premise of ensuring effective targeting of tumor cell killing effect of the lymphocytes, if patients had serious adverse events, lymphocytes anti-EGFR antibody is cleared, thereby improving the safety of the present constructs embodiment of the invention the treatment of cancer patients.

Wherein, independently inventors to achieve the above expression of recombinant receptors are non-functional and optionally at least one of the EGFR receptor following manner:

Carries an internal ribosome entry site sequence (IRES): According to an embodiment of the present invention, the construct further carries an internal ribosome entry site sequence, the internal ribosome entry site sequence SEQ ID NO: 6 shown in FIG. the nucleotide sequence and the internal ribosome entry site sequence arranged in front of said nucleic acid and said nucleic acid encoding a non-functional EGFR in between. Introducing an internal ribosome entry site sequence, such that expression of non-functional EGFR initial coding nucleic acid is not dependent on a 5 'cap structure and a nucleic acid encoding the recombinant receptor and a nucleic acid encoding a non-functional EGFR proportional expression, and thus more conducive expression regulation construct the embodiment of the present invention into a recipient lymphocytes embodiment, the safety of the treatment of the obtained transgenic higher lymphocytes.

Connecting peptide: According to an embodiment of the present invention, the construct may further carry a nucleic acid encoding a linker peptide, the nucleic acid encoding a linker peptide SEQ ID NO: 7 to the nucleotide sequence shown in FIG 10, the connecting peptide 2A is a linker peptide, and a nucleic acid encoding a linker peptide disposed between the nucleic acid and the nucleic acid encoding a non-functional EGFR preceding claim, wherein, SEQ ID nO: 7 encoded a linker peptide having SEQ ID nO: 22 Suo the amino acid sequence shown in, SEQ ID NO: 8 encoding the connecting peptide having SEQ ID NO: 23 amino acid sequence shown in, SEQ ID NO: 9 encoding the connecting peptide having SEQ ID NO: amino acid sequence shown 24, SEQ ID NO : 10 encoding a linker peptide having SEQ ID NO: 25 amino acid sequence shown. According to an embodiment of the present invention, the embodiment of the constructs into the recipient lymphocytes embodiment of the present invention, the encoded peptide can be cut are connected in the lymphocytes, introduction of the linker peptide such that the expressed recombinant receptors and non-functional EGFR expression in a non-confluent lymphocyte membranes.

Promoter: According to an embodiment of the present invention, the construct may further carry a first promoter, the first nucleic acid promoter operatively linked to the aforementioned, optionally, the first promoter They include those selected from CMV, EF-1, LTR or RSV promoter. According to an embodiment of the present invention, the construct further carries a second promoter, said second promoter and said nucleic acid encoding a non-functional EGFR operably linked, optionally, comprise a second promoter selected from since CMV, EF-1, LTR or RSV promoter. The first and second promoter may be activated independently of the above recombinant protein expression of a nucleic acid encoding a non-functional EGFR, and thus more conducive recombinant proteins, regulate the expression of a nucleic acid encoding a non-functional EGFR. The inventors have found, CMV, EF-1, LTR or RSV promoter can efficiently start the above recombinant protein, a nucleic acid encoding a non-functional EGFR recombinant protein expression efficiency of a nucleic acid encoding a non-functional EGFR expression is significantly increased.

Via the internal ribosome entry site sequence, or a first, introduction of a nucleic acid molecule encoding a second promoter or a linker peptide, such that the non-functional EGFR receptor expression efficiently and efficiently expressing the above-described embodiment the recombinant receptors of the present invention transgenic lymphocytes film, and non-functional EGFR receptor and recombinant non-fused receptor-expressing lymphocytes film state, thus ensuring the recombinant protein immunized enhance the biological effect, or effective to achieve the timely removal of transfected lymphocytes, whereby makes lymphocytes targeted killing more significant role in the immune destruction of safety is further improved.

According to an embodiment of the present invention, the vector construct is a retroviral vector, lentiviral vector or an adeno-associated virus vector. The viral vectors of the present invention in embodiments of the packaging and viral infection, a wide range of viral infection, both infected terminally differentiated cells, but also in the infected cell division, either integrated into the host chromosome, can be free in a host outside the chromosome, and thus can achieve a broad spectrum and efficient infection efficiency. Vector described above may be implemented in a high expression of the receptor nucleic acid-carrying cells, high therapeutic efficiency.

According to a particular embodiment of the present invention, to construct a lentiviral vector as an example, the inventor to construct a lentiviral vector, the location of certain viral sequences, the nucleic acid of interest is inserted into the viral genome, resulting in a replication defective virus. In order to produce virions, the inventors have further constructed packaging cell lines (including gag, pol and env genes, but does not include the LTR and packaging components). The inventors recombinant plasmid containing the gene of interest, together with lentivirus LTR and packaging sequences is introduced together with the packaging cell line. Packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, and is then secreted into the medium. Further the inventors collected a matrix comprising a recombinant lentivirus, selectively concentrated, and used for gene transfer. Slow vector can infect a variety of cell types, including dividing cells and can be non-dividing cells.

Further, according to embodiments of the present invention, lentivirus embodiment of the present invention is a composite lentivirus, a lentivirus gene in addition to the common gag, pol and the env, contain other genes and regulatory structural function. Lentiviral vectors are well known to the skilled person, including lentiviruses: human immunodeficiency virus HIV-1, HIV-2 and simian immunodeficiency virus SIV. Lentiviral vectors by using attenuated HIV disease genes produce, such as deleting all the genes env, vif, vpr, vpu and nef, the lentiviral vector biosafety vector form. Recombinant lentiviral vector capable of infecting non-dividing cells, also can be used in vitro and in vivo gene transfer and expression of nucleic acid sequences. For example: in a suitable host cell, and two or more carriers with packaging functions (gag, pol, env, rev and TAT) together capable of infecting non-dividing cells. Targeted recombinant virus is accomplished by an antibody or a specific binding ligand (targeted to a particular cell type receptor) and membrane proteins. Meanwhile, by insertion of a valid targeting sequence (including a regulatory region) to the viral vector of the recombinant virus, along with another gene encoding a ligand for a receptor on a specific target cell, the carrier having a specific targeting. The vector generated various useful lentiviral vectors, and a variety of other methods and operations for changing expressing cells. DNA constructs according to embodiments of the present invention, adeno-associated virus vector of Example (AAV) embodiment of the present invention can be used adenovirus serotypes associated with one or more viral vectors are well known.

Further, according to embodiments of the present invention, an embodiment of the present invention also comprises a minigene. Minigene combination with means (selected nucleotide sequence and operably connected to related sequences necessary) to direct conversion, the expression of transcription and / or gene product in a host cell in vivo or in vitro. Application of the expression "operably linked" sequences comprising the gene control sequence continuously, and trans-acting remote control or control expression of the gene sequence.

Further, the carrier further embodiment of the present invention includes conventional control elements. A large number of expression control sequence (including naturally, inducible and / or tissue specific promoter) may be used. According to an embodiment of the present invention, the promoter is a tissue-specific promoter. According to an embodiment of the present invention, the promoter is an inducible promoter. According to an embodiment, the present invention is a promoter selected from a promoter based on the selected vector. According to an embodiment of the present invention, when selecting a lentiviral vector, a promoter for the CMV IE gene, EF-1α, ubiquitin C, or phosphoglycerate kinase (PGK) promoter. Other conventional expression control sequences include selectable markers or reporter genes include nucleotide sequences encoding geneticin, hygromycin, or puromycin resistance to ampicillin and the like. Other components of the carrier include origins of replication.

Vector construction techniques known to those skilled in the art, such techniques include conventional cloning techniques.

According to an embodiment of the present invention, the inventors constructed an optional non-functional EGFR receptor and recombinant viral vectors of the co-expression of the receptor. Transporting a nucleic acid molecule expression optional embodiment nonfunctional EGFR receptor of the present invention and a recombinant expression plasmid or viral vector receptor is complexed, this viral vector or plasmid or other polymeric materials may be combined to increase its stability, or assist in its targeted motion.

Transfected lymphocytes

Another aspect, the present invention provides a transgenic lymphocytes. According to an embodiment of the present invention, the transgenic lymphocytes expressing recombinant receptors previously described, optionally, said transgenic non-functional lymphocytes express EGFR. Transgenic embodiment of the present invention lymphocytes strong specific killing effect on tumor cells, safety.

According to a particular embodiment of the present invention, the lymphocyte antigen specific T lymphocytes, tumor infiltrating T lymphocytes, peripheral blood T lymphocytes, natural killer T lymphocytes, natural killer cells. The antigen-specific T lymphocytes embodiment of the present invention, tumor-infiltrating T lymphocytes, peripheral blood T lymphocytes, natural killer T lymphocytes or natural killer cells, tumor cells can be achieved specific immune destruction, high safety.

A method of making a transgenic lymphocytes

Another aspect, the present invention provides a process for preparing the foregoing transgenic lymphocytes. According to an embodiment of the present invention, the method comprising: the aforementioned constructs into lymphocytes or T lymphocytes. With the above-described method embodiments of the present invention, it can be conveniently and efficiently obtain transgenic lymphocytes previously described, as described above, the obtained transgenic lymphocytes specific killing of tumor cells in a significant increase of safety.

Therapeutic composition for the treatment of cancer

In another aspect, the present invention provides a therapeutic composition for the treatment of cancer. According to an embodiment of the present invention, the therapeutic composition comprising: a construct previously described, the foregoing transgenic lymphocytes, recombinant receptors or nucleic acid as previously described above. With the embodiment of the therapeutic composition of the present invention enables efficient, safe killing of tumor cells.

According to an embodiment of the present invention, a therapeutic composition embodiments of the present invention provides to a patient, preferably a biocompatible solution is applied to a pharmaceutically acceptable carrier or carriers. As the suspension is prepared in various therapeutic composition or dissolved in a pharmaceutically or physiologically acceptable carrier, such as physiological saline; formulators obvious isotonic saline solution or in another good at this. Suitable carrier depends largely on the administration route. Other aqueous and non-aqueous isotonic sterile injection solutions and sterile water and anhydrous suspension, a pharmaceutically acceptable carrier.

According to an embodiment of the present invention, a sufficient number of the targeted viral vector is transduced into T cells, and to provide sufficient strength transgenic, non-functional expression optionally EGFR receptor and recombinant expression of specific receptors. Doses of therapeutic agents depends on the condition being treated, age, weight and health of the patient, which may cause variability in the patient.

Optionally expression of non-functional EGFR receptor and recombinant expression of the specific receptors for these methods are part of a combination therapy. Performed with these viral vectors and methods for adoptive immunotherapy of anti-tumor T cell, it may be used alone or in combination with other cancer treatment. Under suitable conditions, a method of treatment includes the use of one or more drug therapy.

According to an embodiment of the present invention, the type of cancer is not particularly limited, according to an embodiment of the pharmaceutical composition of the present invention, the effect of PD-L1 specific killing of tumor cells was significantly positive.

Methods to improve the ability of anti-lymphocyte immune

In yet another aspect of the present invention, the present invention provides a method for increasing the ability of anti-lymphocyte immunity. According to an embodiment of the present invention, the method comprising: the recombinant receptor of lymphocyte previously described. With the above-described method embodiments of the present invention can effectively improve the anti-lymphocyte specific immunity to tumor cells.

Methods of treating cancer

In yet another aspect of the present invention, the present invention provides a method of treating cancer. According to an embodiment of the present invention, the method comprising: administering to a patient in front of the recombinant receptor, the nucleic acid according to the foregoing, the previously described transgenic construct or in front of the lymphocytes. With the method of treatment of the present invention, an embodiment can be realized on tumor cells in vivo tumor patients efficiently and safely killing.

Incidentally, the present invention relates to "recombinant receptor" is a recombinant protein or fusion protein, the recombinant receptor expression cell membrane receptors (e.g., lymphocytes) in, receptor protein function, i.e., can specific binding extracellular signaling molecules and activation of a series of biochemical reactions within the cell, the cell generates a corresponding effect on external stimuli.

Below in connection with embodiments of the present invention will be explained.

Those skilled in the art will appreciate that the following examples are only for illustration of the present invention, and should not be construed as limiting the scope of the present invention. Or in the specific technological conditions are not indicated, according to literature techniques or conditions described in the art (see, for example J. Sambrook waiting, "Molecular Cloning A Laboratory Manual", M. Huang Peitang, the third edition embodiment, Science Press) or in accordance with product instructions. The reagents or equipment not specified by the manufacturer, are the conventional products available through the city.

The following cell lines and basic experimental techniques used in the following examples are embodiments:

Generation lentivirus transduced human T-lymphocytes and

Object is to produce replication-defective lentivirus vectors, and lentiviral vectors were collected by centrifugation for transduction of human T lymphocytes.

The following briefly describes Lentiviral vectors collected during experiments: 293T cells were plated in a cell culture dish bottom area of ​​150 square centimeters, and (available from Open Biosystems / Thermo Scientific the description, using the Express-In, Waltham , MA) on 293T cells viral transduction. Cells were added to 15μg per plate lentiviral plasmid, 5 μg of pVSV-G (VSV glycoprotein expression plasmid), 10μg of plasmid pCMVR8.74 (Gag / Pol / Tat / Rev expression plasmid) and 174 μl of Express-In ( at a concentration of 1 μg / μl). Supernatants were collected 24 and 48 hours, and used in ultracentrifuge 28,000 rpm (centrifuge rotor Beckman SW 32Ti, available from Beckman Coulter, Brea, CA) centrifuged for 2 hours under the condition. Finally RPMI-1640 medium 0.75 ml of precipitate was resuspended virus plasmids.

Isolated from volunteer donors primary human T lymphocytes. Human T lymphocytes were cultured in RPMI-1640 medium and CD28 and anti-CD3 monoclonal antibody coated beads (commercially available from Invitrogen, Carlsbad, CA) stimulated activation. 18 to 24 hours after the activation of human T lymphocytes using spin - inoculation of T lymphocytes transduction, transduction as follows: in a 24-well plate, each well lined with 0.5 × 10 6 T lymphocytes, cells added to each well in 0.75 ml of the virus supernatant and resuspended a Polybrene (concentration of 8 μg / ml). A mixture of cells and virus in a tabletop centrifuge plasmid (available from Sorvall ST 40; Thermo Scientific) in centrifugation, centrifugation conditions of room temperature, 2500rpm, 90 minutes. Recombinant human Interleukin -2 (IL-2; available from Novartis, Basel, Switzerland) was added every 2 to 3 days T cell culture medium, the final concentration of IL-2 for the 100-IU / ml, in T lymphocytes in cell culture, cell density was maintained 0.5 × 10 6 ~ 1 × 10 6 / ml. Once transduced T lymphocytes dormant, such as cell growth slows down and becomes smaller cell, wherein the cell growth rate and size by Coulter Counter (available from Beckman Coulter) assessed, or transduced T-lymphocytes at the point of time of a program, T lymphocytes can be used for functional analysis.

Flow cytometer used in the examples of the embodiments of the present application is BD FACSCanto II (available from BD Biosciences), and data flow cytometry using FlowJo version 7.2.5 software (available from Tree Star, Ashland, OR) for analysis.

Cytotoxicity antibody dependent cell-mediated (ADCC)

In the following embodiment, the release assay using 4 hours -51Cr- -EGFR evaluation of the anti EGFR antibody the ability to induce non-functional receptor-dependent cell lysis of lymphocytes expression. Was lentiviral vector transduced human T-lymphocytes were used as target cells. 100μCi Na251CrO4 (available from GE Healthcare Life Sciences, Marlborough, MA) calibration target 2 ~ 5x 106 cells, with the proviso that the calibration shaking for 1 hour at 37 ℃. Cells were rinsed three times with PBS, and resuspended with culture medium (cell density of 1x 105 / ml). Then, cells were plated in the calibrated 96-well plates (plated hole for every 5 × 103 cells, medium was added 50μl) and 50μl of antibody added anti -EGFR (available from Erbitux, Genentech) (final concentration 20μg / ml), pre-conditions at room temperature for 30 minutes. the medium was then replaced with normal media containing the antibody, thereby detecting the spontaneous release of 51Cr. Added to a final concentration of 1% Triton X-100 in order to ensure the maximum release of 51Cr. In the following specific embodiments, the human PBMC (effector cells) were added to plates (5 × 105 per well cells) and the cells incubated overnight at 37 ℃. The next day, cell supernatants were collected and calculated using γ counter in order to determine cpm of 51Cr release. Cell toxicity ratio is calculated using the formula:% specific lysis = (cpm experimental release data - data cpm spontaneous release) / (cpm maximum release data - data cpm spontaneous release) * 100, wherein data is added to the maximum release cpm target cells by triton X-100 implementation, data cpm spontaneous release measured in the absence of anti-EGFR antibody and effector cells.

Chromium release assay

Example 51 using 4-h chromium release assay The cytotoxic activity analysis and evaluation recombinant recipient T cells. Specific steps are as follows: target cells were labeled in the test at 37 ° C for 1 hour 51 Cr. After labeling, run with RPMI medium containing 10% fetal calf serum (FCS) to wash the cells. Run washed, the cells were resuspended in the same medium, cells were resuspended to a concentration of 1 × 10 5 / ml. After transduced T cells at different effector to target cell ratios (E: T) cell suspension was added a test target, and the cells were seeded in 96-well, the volume is 200 microliters per well. The cells were cultured in a 37 ° incubator for 4 hours. After 4 hours, remove 30 microliters of the supernatant from each well and placed in 96-microplate counter counts analysis. Top count NXT analytical instrument is a micro scintillation counter (from Packard Bioscience). All counts the number of holes in the total number of effector cells is calculated based on T cell. Labeled target test PD-L1 cells are positive tumor cells.

Construction of expression vectors co-receptors and non-functional EGFR PD1-CD28-CD3zeta recombinant receptor Example 1

Embodiment, the inventors encoding the extracellular fragment sequence was PD1, CD28 intracellular domain and membrane through ζ- chain sequences cloned T cell receptor composition comprising the lentiviral vector EF-1 promoter (lentiviral vector) of the present embodiment , the process of cloning, restriction selection is digested with XbaI and NotI, and XhoI and NotI double digestion by restriction enzyme digestion, ligation, amplification and filtering of the target plasmid, to generate the receptor expressing recombinant lentivirus plasmid (LV-PD1-CD28-CD3ζ). IRES and expression sequences comprising non-functional EGFR receptor was cloned into the LV-PD1-CD28-CD3ζ plasmid, constructed LV-PD1-CD28-CD3ζ-tEGFR. 1 is a schematic lentiviral vectors (wherein E represents the extracellular segment; tEGFR represents non-functional EGFR), comprising the sequence encoding the PD1-CD28-CD3ζ recombinant receptor, IRES, and a sequence encoding a non-functional EGFR receptor. Sequence encoding PD1-CD28-CD3ζ recombinant receptors under control of a promoter promoter of EF-1, EGFR receptor expression of non-functional sequence of the mRNA transcript as a separate unit from an IRES sequence after the translation start.

Further, it was PD1 encoding the extracellular and transmembrane fragment sequence segment, sequences within the CD28 intracellular zeta-chain segment and T cell receptor composition was cloned into the lentiviral vector containing the process of EF-1 promoter described above.

Example 2 anti-EGFR antibody may be cleared effectively kill T lymphocytes co-express non-functional receptors EGFR receptor and recombinant

In the present embodiment, the peripheral blood lymphocytes from the blood supply does not anonymous. Peripheral blood lymphocytes were separated by gradient centrifugation, a centrifuge is a gradient Ficoll-Hypaque. T lymphocytes and T cell activating factor beads CD3 / CD28 (commercially available from Invitrogen, Carlsbad, CA) were incubated at 5% CO2,37 degrees Celsius for 72 hours the medium was supplemented with 2mmol / L glutamine, 10% high temperature inactivated fetal calf serum (the FCS) (available from Sigma-Aldrich Co.) RPMI 1640 medium and 100U / ml penicillin / streptomycin double antibody (available from Invitrogen Gibco Cat.no.12633-012). 72 hours after activation, cells were rinsed with wash, wash away magnetic beads. The T cells were seeded in lined with a recombinant fibronectin fragment (FN ch-296; Retronectin) on the cell culture dish, and lentiviral transduction, transduction lentivirus were LV-PD1-CD28-CD3ζ-tEGFR as, LV-PD1 -CD28-CD3ζ or load (LV-GFP) transduction as previously described. Expression of non-functional EGFR receptor T cells after transduction with anti-EGFR antibody staining, cells and flow cytometry (FACS) to be separated, isolated T cells were cultured in RPMI-1640 medium and IL-2 using recombinant human factor (100 ng / ml; purchased from R & D Systems) to induce amplification of 7-10 days, and then used as target cells in the experiments. The inventors measured the killing effect of different lentivirus transduced T cell-mediated anti-EGFR antibody-specific ADCC assay by measurement methods using standard chromium release assay 4- 51 hours, 4 hours 51 Cr release assay as described in Example 1 the. The results shown in Fig. As shown, the anti-EGFR antibody may be effective killing mediated co-expression of two heterologous PD1-CD28-CD3ζ recombinant receptors and non-functional EGFR receptor of T lymphocytes, but not anti-EGFR antibody-mediated expression of heterologous killing only PD1-CD28-CD3ζ recombinant T cell receptors, anti-EGFR antibody is not heterologous anti-idling lentivirus-mediated transduction of T lymphocytes, statistical data representing three apertures mean ± SEM.

Example 3 EGFR co-expression of non-functional receptors and the ability of tumor cytolytic T lymphocytes PD1-CD28-CD3ζ recombinant receptor

In the present embodiment, the peripheral blood lymphocytes isolated by gradient centrifugation, a centrifuge is a gradient Ficoll-Hypaque. T lymphocytes and T cell activating factor beads CD3 / CD28 (commercially available from Invitrogen, Carlsbad, CA) were incubated at 5% CO2,37 degrees Celsius for 72 hours the medium was supplemented with 2mmol / L glutamine, 10% high temperature inactivated fetal calf serum (the FCS) (available from Sigma-Aldrich Co.) RPMI 1640 medium and 100U / ml penicillin / streptomycin double antibody (available from Invitrogen Gibco Cat.no.12633-012). 72 hours after activation, cells were rinsed with wash, wash away magnetic beads. The T cells were seeded in lined with a recombinant fibronectin fragment (FN ch-296; Retronectin) on the cell culture dish, and lentiviral transduction, transduction lentivirus were LV-PD1-CD28-CD3ζ-tEGFR as, LV-PD1 -CD28-tEGFR (shown in Figure 1), LV-tEGFR (shown in Figure 1), or no load (LV-GFP), transduction processes previously described. After transduction and T cell cultures (100ng / ml; purchased from R & D Systems) with recombinant human IL-2 in RPMI-1640 medium factor induced amplification in 7-10 days and then subjected to functional testing experiments. The inventors measured various lentivirus transduced T cells of PD-L1-positive glioma cell killing effect, the ratio of effector to target cells 10: 1 or 25: 1 or 50: 1, using standard methods of measurement 4- hour 51 Cr release assay, wherein 4-hour 51 Cr release assay as previously described.

The test results shown in Figure 3, Figure 3 show the results: co-expression of receptor PD1-CD28-CD3ζ and non-functional EGFR receptor lentivirus transduced T lymphocytes (LV-PD1-CD28-CD3ζ-tEGFR T lymphocytes ) can be significantly killing brain tumor cells of PD-L1 +. PD1-CD28 co-expression of brain receptors (not CD3zeta chain fragment) and non-functional EGFR receptor lentivirus transduced T lymphocytes (LV-PD1-CD28-tEGFR T lymphocyte) had no significant anti-PD-L1 + a the role of tumor cells. Express only non-functional EGFR receptor lentivirus transduced T lymphocytes (LV-tEGFR T lymphocytes) or load lentivirus transduced T lymphocytes (control LV-GFP T lymphocytes) to PD-L1 + Brain no tumor cells in vitro.

In the description of the present specification, reference to the term "one embodiment," "some embodiments", "an example", "a specific example", or "some examples" means that a particular feature of the described embodiments or examples described , structure, material, or characteristic is included in at least one embodiment of the present invention, embodiments or examples. In the present specification, a schematic representation of the above terms must not be the same for the embodiment or exemplary embodiments. Furthermore, the particular features, structures, materials, or characteristics described may be in any one or more embodiments or examples combined in suitable manner. Furthermore, different embodiments or examples and embodiments or features of different exemplary embodiments without conflicting, those skilled in the art described in this specification can be combined and the combination thereof.

Although the above has been illustrated and described embodiments of the present invention, it is understood that the above embodiments are exemplary and are not to be construed as limiting the present invention, those of ordinary skill in the art within the scope of the present invention may be the above-described Example changes, modifications, substitutions and variations.

Claims (22)

  1. A recombinant receptor, characterized by comprising:
    Checkpoint immune molecule fragment;
    Immunostimulatory molecule fragment; and
    T cell receptor zeta chain.
  2. The recombinant receptor according to claim 1, wherein said molecule is an immune checkpoint PD1.
  3. The recombinant receptor according to claim 2, wherein said immune checkpoint molecular fragment comprising an extracellular region PD1 transmembrane region, and optionally, the immunostimulatory molecule comprises a fragment of the CD28 intracellular region and any selected transmembrane region.
  4. The recombinant receptor according to claim 3, characterized in that, comprising:
    (A) said PD1 extracellular and transmembrane domains; and
    (B) the CD28 intracellular region,
    Or include:
    (I) the extracellular region PD1; and
    (Ii) said CD28 intracellular and transmembrane domains.
  5. The recombinant receptor according to claim 1, wherein the T cell receptor zeta chain CD3zeta chain.
  6. The recombinant receptor according to claim 1, characterized in that, connected to the C-terminus of the molecule fragment immune checkpoint immunostimulatory molecule with the N-terminal fragment, the immunostimulatory molecule fragments of the C-terminus of T connected to the N-terminal cell receptor zeta chain.
  7. A recombinant receptor, wherein said recombinant receptor SEQ ID NO: 1 or the amino acid sequence shown in 2.
  8. A nucleic acid comprising a recombinant receptor according to any of claims 1 to 7, the nucleic acid encodes claim,
    Optionally, the nucleic acid having SEQ ID NO: 3 or the nucleotide sequence shown 4.
  9. A construct wherein the construct carrying the nucleic acid of claim 8.
  10. The construct according to claim 9, wherein the nucleic acid further encoding a non-functional EGFR carrying the construct, said nucleic acid encoding a non-functional EGFR having SEQ ID NO: 5 nucleotide sequence.
  11. The construct according to claim 10, wherein said construct further carries an internal ribosome entry site sequence, the internal ribosome entry site sequence SEQ ID NO: 6 nucleotide sequence and the internal ribosome entry site between the sequence of a nucleic acid is provided in the claims of non-functional EGFR, and a nucleic acid encoding said 8.
  12. The construct according to claim 10, wherein said construct further carry a nucleic acid encoding a linker peptide, the nucleic acid encoding a linker peptide SEQ ID NO: 7 to 10, the nucleotide sequence, and nucleic acid encoding a linker peptide is disposed between a nucleic acid as claimed in claim EGFR nonfunctional nucleic acids and the encoded 8.
  13. The construct according to claim 9, wherein said construct further carrying a first promoter, the first nucleic acid of claim 8 promoter operatively linked to the claims,
    Optionally, the first promoter selected from the group comprising CMV, EF-1, LTR or RSV promoter.
  14. The construct according to claim 10, wherein said construct further carries a second promoter, said second promoter and said nucleic acid encoding a non-functional EGFR operably linked,
    Optionally, the second promoter selected from the group comprising CMV, EF-1, LTR or RSV promoter.
  15. The construct according to claim 9, characterized in that the carrier body is a retrovirus vector, lentivirus vector or an adeno-associated virus vector the construct.
  16. A construct wherein the construct carrying the nucleic acid molecule of the following:
    (1) a nucleic acid molecule encoding a fragment of such molecule immune checkpoint, the checkpoint immune molecule fragment having SEQ ID NO: 11 or the amino acid sequence shown in FIG. 12, the nucleic acid molecule encoding an immune checkpoint molecule fragments having SEQ ID NO: the nucleotide sequence shown in 1314;
    Nucleic acid molecule (2) a fragment encoding an immunostimulatory molecule, the immunostimulatory molecule fragment having SEQ ID NO: 15 or the amino acid sequence set forth in 16, said nucleic acid molecule encoding an immunostimulatory molecule fragments having SEQ ID NO: 18. 17 or nucleotide sequence; and
    (3) a nucleic acid molecule encoding the T cell receptor zeta chain of the T cell receptor zeta chain having SEQ ID NO: 19 amino acid sequence of the nucleic acid molecule of the T cell receptor zeta chain encoding a SEQ ID NO : 20 is the nucleotide sequence shown,
    Optionally, the construct further carry a nucleic acid molecule encoding a non-functional EGFR, wherein the non-functional EGFR encoding nucleic acid molecule having SEQ ID NO: 5 nucleotide sequence, the non-functional EGFR having SEQ ID NO : 21 amino acid sequence shown,
    Optionally, the construct further carries an internal ribosome entry site sequence of a nucleic acid molecule, said internal ribosome entry site sequence of a nucleic acid molecule having SEQ ID NO: 6 nucleotide sequence, and the encoding the internal ribosome entry site sequence of a nucleic acid molecule is provided between the non-functional EGFR nucleic acid molecule and said nucleic acid molecule encoding said encoding T cell receptor zeta chain,
    Optionally, the construct further carry a nucleic acid encoding a linker peptide, the nucleic acid encoding a linker peptide SEQ ID NO: 7 to 10, the nucleotide sequence of the connecting peptide has SEQ ID NO: 22 is ~ amino acid sequence shown between 25 and nucleic acid encoding a linker peptide is provided to the T cell receptor zeta chain coding nucleic acid molecule encoding a non-functional EGFR and said nucleic acid molecule,
    Optionally, the construct further carrying a first promoter, the first promoter and said nucleic acid molecule encoding a promoter immune checkpoint operably linked molecule fragments,
    Optionally, the first promoter selected from the group comprising CMV, EF-1, LTR or RSV promoter,
    Optionally, the construct further carries a second promoter, said second promoter and said nucleic acid encoding a non-functional EGFR molecule is operably linked,
    Optionally, the second promoter selected from the group comprising CMV, EF-1, LTR or RSV promoter,
    Optionally, the carrier body is a retrovirus vector, lentivirus vector or an adeno-associated virus vector the construct.
  17. A transgenic lymphocytes, wherein the lymphocytes express a recombinant receptor transgene according to any one of claims 1 to 7,
    Optionally, the transgenic non-functional lymphocytes express EGFR.
  18. The transgenic lymphocytes in claim 17, wherein said lymphocyte antigen specific T lymphocytes,
    Optionally, the tumor infiltrating lymphocytes are T-lymphocytes,
    Optionally, the lymphocytes are peripheral blood T lymphocytes,
    Optionally, the lymphocyte is a Natural Killer T lymphocytes,
    Optionally, the natural killer cells are lymphocytes.
  19. A method for the preparation of any of claims 17 to 18, one of the transgenic lymphocytes, characterized by comprising:
    To any of claims 9 to 16, one of the construct into lymphocytes or T lymphocytes.
  20. A therapeutic composition for treating cancer, characterized by comprising:
    The recombinant receptor according to any one of claims 1 to 7, a nucleic acid according to claim 8, according to any one of claim 9 to 16, construct or transgene of claims lymphatic according to any one 17 to 18 cell.
  21. A method for treating immune lymphocyte cytolytic activity increase, characterized by comprising:
    Lymphocytes expressing the recombinant receptor claimed in claim any one of claims 1 to 7.
  22. A method for treating cancer, characterized by comprising:
    Administering to a patient as claimed in claim recombinant receptor according to any one of claims 1 to 7, a nucleic acid according to claim 8, 9 to any one of claims 16 or construct as claimed claim any one of claims 17 to 18 the transfected lymphocytes.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103965361A (en) * 2013-02-06 2014-08-06 上海细胞治疗工程技术研究中心有限公司 Single chimeric converter for T-cell signal and application thereof
CN104769103A (en) * 2012-09-04 2015-07-08 塞勒克提斯公司 Multi-chain chimeric antigen receptor and uses thereof
WO2016014565A2 (en) * 2014-07-21 2016-01-28 Novartis Ag Treatment of cancer using humanized anti-bcma chimeric antigen receptor
WO2016025880A1 (en) * 2014-08-14 2016-02-18 Novartis Ag Treatment of cancer using gfr alpha-4 chimeric antigen receptor
CN105392888A (en) * 2013-03-16 2016-03-09 诺华股份有限公司 Treatment of cancer using humanized anti-cd19 chimeric antigen receptor
WO2016070061A1 (en) * 2014-10-31 2016-05-06 The Trustees Of The University Of Pennsylvania Methods and compositions for modified t cells

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104769103A (en) * 2012-09-04 2015-07-08 塞勒克提斯公司 Multi-chain chimeric antigen receptor and uses thereof
CN103965361A (en) * 2013-02-06 2014-08-06 上海细胞治疗工程技术研究中心有限公司 Single chimeric converter for T-cell signal and application thereof
CN105392888A (en) * 2013-03-16 2016-03-09 诺华股份有限公司 Treatment of cancer using humanized anti-cd19 chimeric antigen receptor
WO2016014565A2 (en) * 2014-07-21 2016-01-28 Novartis Ag Treatment of cancer using humanized anti-bcma chimeric antigen receptor
WO2016025880A1 (en) * 2014-08-14 2016-02-18 Novartis Ag Treatment of cancer using gfr alpha-4 chimeric antigen receptor
WO2016070061A1 (en) * 2014-10-31 2016-05-06 The Trustees Of The University Of Pennsylvania Methods and compositions for modified t cells

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